Cells were washed by phosphate-buffered saline (PBS) on ice, then scraped using a cold plastic cell scraper, and the cell suspension was gently transferred into a pre-cooled microcentrifuge tube with lysis buffer. Next, the cells were centrifuged at 12,000 rpm at 4 °C for 20 min. The protein concentration was determined by bicinchonic acid (BCA) disodium salt (ab146331, Abcam). Next, 20 µg of total protein was loaded into the SDS-PAGE gel wells (10%), and the electrophoresis were performed at 90 V for 2 h. Then, protein was transferred from the gel to the membrane. For ZEB2 analysis, the membrane was blocked at 4 °C overnight in blocking buffer, and washed by tris-buffered saline with Tween20 (TBST). After this, the membrane was incubated with ZEB2 antibody (cat.no, ab138222, Abcam, Cambridge, UK) at a dilution of 1:1,000 at room temperature for 2 h. Then, the whole membrane was incubated by the secondary antibody immunoglobulin G (IgG) (ab7090, 1:2,000 dilution, Abcam) at 4 °C overnight. After being washed by TBST three times, the membrane was mixed with ECL Western Blotting Substrate kit (cat.no, ab65623, Abcam) at a 1:1 ratio, incubated for 3 min, and get the blot in a X-ray film.
Ab146331
Manufactured by Abcam
Sourced in United States
Ab146331 is a recombinant monoclonal antibody that targets an undisclosed antigen. It is produced in a mammalian expression system.
Automatically generated - may contain errors
2 protocols using ab146331
1
Western Blot Analysis of ZEB2 Protein
2
Western Blot Analysis of cAMP, PKA, and CREB Signaling
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The mouse cortex and hippocampal tissue were rapidly isolated on ice, total proteins were extracted, the protein concentration was determined by the BCA reagent total proteins were extracted and the concentration was determined by BCA reagent (Abcam, USA, ab146331). The samples were subjected to SDS-PAGE electrophoresis, and the separated proteins were transferred to PVDF membranes. The blots were blocked with 5% skim milk at room temperature for 1 h, and then the membrane was incubated with the primary antibody overnight at 4° C, including anti-cAMP (Abcam, USA, ab76238, 1:2000), anti-PKA (catalytic subunits) (Abcam, USA, ab59218, 1:1000), anti-CREB (Abcam, USA, ab32515, 1:500). Then, the cells were incubated with horseradish peroxidase conjugated goat anti-rabbit IgG H&L (Abcam, USA, ab6721,1:5000) for 2 h at room temperature. After incubation with a properly titrated secondary antibody, the cells underwent ECL luminescence development. The developed image was scanned to a computer for analysis after exposure.
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